The present invention is directed to macrocapsules and their use as implantation and retrieval devices for cells.
The methods of implanting living cells and or tissue into the body to provide therapeutically useful substances (cell therapy) is currently the focus of a number of research efforts. Among others, therapeutic applications for cell therapy have been suggested in the areas of diabetes and neural degenerative diseases such as Alzheimer""s Disease, Parkinson""s Disease and epilepsy. Additionally, cells have also been shown to have great therapeutic potential for the removal of detrimental substances from the body. For example, hepatocytes have been implanted for the treatment of high cholesterol levels as shown by Wang et al., Transplantation Proceedings, 23:894-895 (1991).
In one form of cell therapy, the cells that are implanted into the patient have been genetically modified (transduced) in vitro with exogenous genetic material so as to enable the cells to produce a desired biological substance that is useful as a therapeutic agent. There are variety of mechanisms for transducing cells. Retroviral vectors have been of interest due to the ability of some vectors to transduce a very high percentage of target cells. Replication of target cells is necessary for proviral integration to occur using these vectors. In theory, cells transduced with retroviruses are not able to spread virus to other cells. However, several safety issues surround the use of these systems. Risks include the potential during production of the retroviral vector preparations for contamination with infectious viruses or pathogens (Cornetta, Human Gene Therapy Vol 2:5-14, 1991).
Adenoviruses are also used as gene transfer vectors. They are double stranded DNA viruses capable of infecting post-mitotic cells, and their successful transfection of host cells can result in the expression of large amounts of gene product. Adenoviruses are a minor pathogen in humans and are not normally associated with malignancies. While the adenovirus vector generally remains episomal and does not undergo replication, studies have shown that gene expression can persist for a significant period, opening up the possibility that the vectors are at least replication-competent.
While present cell therapy methods show great therapeutic potential, they have several limitations. The injection of cells into a patient can create a severe immune reaction. An immune reaction to a first injection of cells will most likely preclude a second injection, thus limiting the benefit which can be gained from such treatment. Some candidate cell types may shed virus particles which could be detrimental to the host and therefore may not be considered suitable for implantation. In the case of cells that have been genetically modified to produce a desired biological substance using viral techniques, the cells may harbor viral particles, thus allowing the possibility of infecting a patient""s cells. Another disadvantage with the current cell therapy methods is that many cells that might be suitable for such therapies are known to migrate in situ, (e.g. glial cells). The inability to retain implanted cells in a fixed location may make them unsuitable as therapeutic agents. Likewise, cells which are autologous or even allogeneic to the host may continue to divide unchecked and produce tumors.
Current methods of cell therapy do not readily allow termination of, or adjustments to, the cell therapy protocol once the cells are implanted. This is because cells implanted into a patient""s body are not well isolated from the patient""s own tissue and thus cannot be readily retrieved or manipulated. This creates a real fear when the cells have been genetically modified using retroviral particles because implanted cells could migrate in situ and potentially result in proviral integration into the host germ line cells, thus passing the provirus onto offspring.
There are a number of specific circumstances where it may be critical to terminate or remove implanted therapeutic cells. These include:
1. One or more of the implanted cells has become oncogenic or tumor forming, or has induced an adverse immune reaction.
2. A dose adjustment may require a reduction in the number of implanted cells at specific times.
3. Genetically engineered cell populations may have a small number of cells with improper incorporation of genetic material leading to detrimental effects.
4. The therapy may have a defined end point (e.g. growth hormone treatment) at which the point the continued presence of the therapeutic cells is unwanted or detrimental.
5. Genetically modified cells may develop an adverse response to concurrently administered pharmacological agents.
6. The development of an improved therapeutic option may warrant a change in therapy requiring removal of the transplanted cells.
Incorporation of inducible suicide genes into cells for implantation has been suggested as a potential means to terminate cell therapy i.e. by killing the implanted cells. One disadvantage of this approach, however, is that since cell division is a stochastic process, there is always a chance that the suicide mechanism in one or a few cells might become inactivated. Undesirable effects could be produced even if only one of the cells continued to divide. This approach also may have undesirable results caused by the dumping of secretory products such as growth factors or proteases or the release of viruses, etc., due to the large scale destruction of implanted cells.
Cell encapsulation methods have been used to isolate cells while allowing the release of desired biological materials. Two techniques have been used, microencapsulation and macroencapsulation. Typically, in microencapsulation, the cells are sequestered in a small permselective spherical container, whereas in macroencapsulation the cells are entrapped in a larger non-spherical membrane.
Lim, U.S. Pat. Nos. 4,409,331 and 4,352,883, discloses the use of microencapsulation methods to produce biological materials generated by cells in vitro, wherein the capsules have varying permeabilities depending upon the biological materials of interest being produced. Wu et al, Int. J. Pancreatology, 3:91-100 (1988), disclose the transplantation of insulin-producing, microencapsulated pancreatic islets into diabetic rats. Aebischer et al., Biomaterials, 12:50-55 (1991), disclose the macroencapsulation of dopamine-secreting cells.
Various polymeric materials have been used in the art for filtering viruses from liquids. Anazawa et al. (U.S. Pat. No. 5,236,588) teach an ultrafiltration membrane prepared by irradiating a monomer to produce a membrane having communicating pores. Allegrezza U.S. Pat. No. 5,096,637) reports an asymmetric composite ultrafiltration membrane for isolating viruses from protein containing solutions. DiLeo (U.S. Pat. No. 5,017,292) teaches asymmetric skinned membranes comprising a porous substrate, an ultrafiltration surface skin and an intermediate zone free of voids which form a break in the skin and which cause fluid to communicate directly with the porous substrate. The membranes are used for isolating viruses from protein containing solutions. U.S. Pat. Nos. 4,808,315 and 4,857,196 teach a hydrophilic hollow fiber membrane for removing virus from a protein containing solution.
None of the above references describe virally retentive, permselective, biocompatible membranes that can be implanted into a patient for cell-therapy purposes. There are many situations where it would be useful to provide cell therapy for the in vivo delivery of biological materials, while substantially preventing release of detrimental viruses that may be shed from the encapsulated cells. There are also many situations where easy termination of cell therapy treatment would be desirable. However, effective means for such treatments are lacking in the art of cell therapy.
Accordingly, it is an object of the present invention to provide methods for cell therapy that can be easily terminated at a desired time by providing a method for the retrieval of implanted cells.
It is another object of the present invention to provide macrocapsules to encapsulate cells wherein the capsules have selected permeability characteristics based upon their particular usage and desired viral retentivity characteristics.
It is another object of the present invention to provide methods for testing the viral retentivity of macrocapsules.
These and other objects and features of the invention will be apparent to those skilled in the art from the following detailed description and appended claims.
None of the foregoing references is believed to disclose the present invention as claimed and is not presumed to be prior art. The references are offered for the purpose of background information.
An implantable permselective macrocapsule is described for use in cell therapy. The macrocapsule comprises a core comprising living cells that are capable of secreting a selected biologically active product or of providing a selected biological function and an external jacket which surrounds the core. The jacket comprises a biocompatible material that is substantially free of the encapsulated cells and has a nominal molecular weight cutoff sufficient to retain the cells within said macrocapsule. In some embodiments the nominal molecular weight cutoff of the macrocapsule is below the molecular weight of detrimental viruses which may be shed from said living cells.
A method for determining the viral retentivity of an external jacket of an implantable permselective macrocapsule is also described wherein the external jacket comprises a hollow fiber and virus is loaded into the hollow fiber. The ends of the hollow fiber are then sealed and the fiber is placed into a bath solution. After a time interval, the bath solution is innoculated onto a lawn of bacteria susceptible to the virus and viral retentivity is calculated based on the number of plaques formed.